CD45 (Protein tyrosine phosphatase, PTPRC)

CD45 (cluster of differentiation 45), also known as protein tyrosine phosphatase, receptor type C (PTPRC) or leukocyte common antigen (LCA), is a transmembrane receptor-type tyrosine phosphatase expressed on the surface of all differentiated hematopoietic cells. The phosphatase activity of CD45 antigen is necessary for a variety of cellular processes including efficient T- and B-cell antigen receptor mediated signal transduction, cell growth, differentiation and oncogenic transformation. Organisms lacking CD45 are severely immune-deficient and are often characterized by few T cells in periphery, as well as, impaired T- and B-cell responses.

Various members of the Src family of tyrosine kinases have been identified as the main substrates for CD45 phosphatase activity, and CD45 has been shown to function as both positive and negative regulators of Src family members. In T cells, the tyrosine kinase Lck is dephosphorylated by CD45 proteins to promote cytokine production and T cell proliferation. Alternatively, CD45 can function as a Janus kinase (JAK) phosphatase, negatively regulating cytokine receptor activation involved in the differentiation, proliferation and anti-viral immunity of hematopoietic cells. In flow cytometric immunophenotyping, antibodies against CD45 have been extensively used as a phenotypic marker for the differentiation of immune cell types, particularly in the study of cell biology, immunology and neuroscience.

CD45 Structure

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CD45, which comprises ~10% of all lymphocyte surface proteins, is a type I transmembrane protein encoded by a 35 exon-containing PTPRC gene located on chromosome 1. It consists of a large, heavily glycosylated extracellular domain (390 - 550 amino acids) and an extensive cytoplasmic tail (~700 amino acids), which consists of two domains with phosphatase homology. The proximal cytoplasmic domain of CD45 has phosphatase activity, whereas the second domain does not. The second domain, however, is required for proper folding and substrate recruitment.

The CD45 extracellular domain has an overall rod-like structure that projects from the cell, as well as folds and bends laterally. The extracellular domain contains three membrane proximal fibronectin type III repeats, a cysteine rich region and the variable N-terminal region. The fibronectin type III repeats serve as a platform for N-glycosylation sites and maintains CD45 structural integrity. The N-terminal region, which is heavily glycosylated, contains a number of domains dependent on alternative splicing of exons 4 to 7 of the CD45 gene.

Human CD45 isoforms - RA, RO, RB, RAB, RBC and RABC

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As a result of alternative splicing, human CD45 can exist as six different isoforms - RA, RO, RB, RAB, RBC and RABC - which vary in size from 180 to 220 kDa and differ in the exon composition of their extracellular domains. All of the isoforms contain the same eight amino acids at their amino terminus followed by either an A, B or C peptide, or some combination of the three. The expression of different CD45 isoforms depends on the state of activation and differentiation of hematopoietic cells, and it has been used to distinguish subsets of different T cell populations. For instance, immature CD4⁺CD8⁺ thymocytes mainly express the low molecular-weight isoforms, whereas mature CD4⁺ and CD8⁺ thymocytes and peripheral T cells can express multiple isoforms.

CD45 Antibodies

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Monoclonal CD45 antibodies recognize all isoforms of human CD45 and are frequently used in flow cytometry and immunofluorescence applications to identify immune cell subsets expressing CD45. Cells known to express CD45 surface markers include T cells, B cells, dendritic cells, NK cells, Stem cell/precursors, macrophages, monocytes and granulocytes.

AAT Bioquest offers a comprehensive catalog of CD45 antibodies purified by affinity chromatography and conjugated to a variety of fluorophores under optimal conditions that minimize unconjugated fluorophore and antibody. Available fluorophores include:

• iFluor® dyes - bright, photostable dyes with optimized flow cytometry (FACS), fluorescence imaging and in vivo imaging applications.
• mFluor™ dyes - bright, photostable dyes with optimized for flow cytometry (FACS) applications.
• Alexa Fluor^® dyes - suitable for flow cytometry (FACS) and fluorescence imaging applications.
• Classic dyes - suitable for flow cytometry (FACS) and fluorescence imaging applications.
• Phycobiliproteins and Tandem dyes - intensely bright dyes for flow cytometry (FACS) and multiparametric analysis.

1. FC = Flow Cytometry; ELISA = Enzyme-linked immunosorbent assay; IF = Immunofluorescence; IHC-F = Immunohistochemistry (Frozen); WB = Western Blot.

iFluor® Dyes Labeled to CD45 Antibodies

The following table outlines the fluorescence properties of available iFluor® dye labeled anti-human CD45 antibodies for use in flow cytometry (FACS) and fluorescence imaging applications. Conjugates made with iFluor® dyes exhibit superior brightness and photostability, outperforming Alexa Fluor^® conjugates and other spectrally similar conjugates. For additional information on iFluor® dye-labeled CD45 antibodies and availability of other clones click on any label in the table below.

1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).li>
2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).
3. CF at 260 nm is the correction factor used for eliminating the dye contribution to the absorbance at 260 nm (for oligo and nucleic acid labeling).
4. CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptides and protein labeling).

mFluor™ Dyes Labeled to CD45 Antibodies

The following table outlines the fluorescence properties of available mFluor™ dye labeled anti-human CD45 antibodies for use in flow cytometry (FACS). Each mFluor™ dyes is optimally excited by one of the major laser lines commonly equipped in flow cytometers, such as the 405 nm, 488 nm, 532 nm, 561 nm or 633 nm laser lines. For additional information on mFluor™ dye-labeled CD45 antibodies and availability of other clones click on any label in the table below.

1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).
2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).>
3. CF at 260 nm is the correction factor used for eliminating the dye contribution to the absorbance at 260 nm (for oligo and nucleic acid labeling).
4. CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptides and protein labeling).

Alexa Fluor^® Dyes Labeled to CD45 Antibodies

The following table outlines the fluorescence properties of available Alexa Fluor^® dye labeled anti-human CD45 antibodies for use in flow cytometry (FACS) and fluorescence imaging applications. For additional information on Alexa Fluor^® dye-labeled CD45 antibodies and availability of other clones click on any label in the table below.

1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).
2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).
3. CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptides and protein labeling).

Classic Dyes Labeled to CD45 Antibodies

The following table outlines the fluorescence properties of available classic dye labeled anti-human CD45 antibodies for use in flow cytometry (FACS) and fluorescence imaging applications. For additional information on classic dye-labeled CD45 antibodies and availability of other clones click on any label in the table below.

1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).
2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).
3. CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptides and protein labeling).

PE, APC, PerCP and Tandem Dyes Labeled to CD45 Antibodies

The following table outlines the fluorescence properties of available phycoerythrin (PE), allophycocyanin (APC), PerCP and tandem dye labeled anti-human CD45 antibodies for use in flow cytometry (FACS). Phycobiliproteins are uncharacteristically bright due to their high molar extinction coefficients and quantum yields, an enviable quality when imaging low-abundance targets. However, since phycobiliprotiens photobleach rapidly, they are not recommended for microscopy. For additional information on phycobiliprotein-labeled CD45 antibodies and availability of other clones click on any label in the table below.

1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).

References

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1. Dawes R, Petrova S, Liu Z, Wraith D, Beverley PC, Tchilian EZ. Combinations of CD45 isoforms are crucial for immune function and disease. J Immunol. 2006;176(6):3417?3425. doi:10.4049/jimmunol.176.6.3417
2. Desai DM, Sap J, Silvennoinen O, Schlessinger J, Weiss A. The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation. EMBO J. 1994;13(17):4002?4010.
3. Hermiston ML, Xu Z, Weiss A. CD45: a critical regulator of signaling thresholds in immune cells. Annu Rev Immunol. 21, 107-137.
4. Rossy J, Williamson DJ, Gaus K. How does the kinase Lck phosphorylate the T cell receptor? Spatial organization as a regulatory mechanism. Front Immunol. 2012;3:167. Published 2012 Jun 19. doi:10.3389/fimmu.2012.00167